Genital lesions caused by herpes simplex virus 2 (HSV-2) are a source of virus transmission to newborns and a portal of entry for HIV, but despite its medical importance, HSV-2 is understudied. The HSV virion contains tegument proteins that regulate viral and host cell processes. The tegument proteins UL13 and virion host shutoff (vhs) are non-essential in culture but affect HSV replication and pathogenesis. The basis for the in vivo attenuation of UL13 mutants is not known. Our data indicate that HSV-2 vhs interferes with the type I interferon (IFN) response in vivo, including phosphorylation of the PKR-substrate eIF2alpha, but the mechanism of interference remains to be elucidated. Published studies link the UL13 viral kinase with the synthesis and/or function of vhs, which could in turn affect IFN sensitivity, but the significance in the virus life cycle of this relationship is not understood. Because tegument proteins play structural roles in the virion, viruses expressing full-length UL13 or vhs proteins containing point mutations that abrogate a specific function rather than viruses lacking these proteins would be ideal for discerning their role in pathogenesis. Using homologous recombination, we will construct mutant viruses with defined point mutations to investigate the roles of UL13 and vhs in vivo using a mouse model of HSV-2 genital infection. We hypothesize that UL13 regulates phosphorylation, synthesis and function of several HSV proteins that influence virus virulence, including vhs, and that HSV-2 vhs interferes with the type I IFN response. Aim 1: We will determine the functional implications of HSV-2 UL13 kinase activity by comparing replication and virulence of a UL13 kinase activity mutant to wild-type HSV-2 in vivo, and we will dissect the reasons for attenuation in vitro. Aim 2: We will determine the basis for HSV-2 vhs-mediated type I IFN resistance using a vhs RNAse activity mutant, and we will determine the role of vhs inhibition of the PKR antiviral pathway in resistance. Aim 3: We will investigate the type I IFN sensitivity of UL13 mutant virus and determine whether sensitivity is due to an effect of UL13 on vhs. These investigations will improve our understanding of the roles of the UL13 and vhs enzymes in HSV-2 pathogenesis.